Envelope glycoprotein (Env) spikes are heterogeneous, labile and low in copy number on HIV-1 virions. These properties appear to contribute to poor (i.e. predominantly non-neutralizing) antibody responses to primary HIV-1, particularly early in acute natural infection. This project is to develop covalently tethered HIV spikes of high stability, structural homogeneity and immunogenicity in an attempt to elicit superior HIV neutralizing antibody responses in animals. HIV-1 spikes, natively displayed on virion particles, will be covalently trapped in fully cleaved and functionally relevant conformations. A panel of small molecule tethers functionalized with chemistry able to ligate lysine, tyrosine or arginine side chains, or to crosslink carbohydrate moieties, will be screened to stabilize mature, homogeneously trimeric Env spikes. The products will be profiled for stability, homogeneity, antigenicity and production yield, the latter following Env purification and immunodepletion using neutralizing and non-neutralizing antibody. Relationships between the parameters described and other physical, structural and antigenic characteristics of tethered Env spikes will be analyzed, as well as Env strain and sequence assessed in order to identify leads for immunization. Covalent trapping of receptor-activated Env spikes using novel chemistries will also be investigated. The most robust and antigenically native tethered Env spikes will be purified and used to immunize animals and serum antibody binding specificity and neutralization titers will be determined. Empirical immunization data will be used to advance candidate(s) for confirmatory immunization studies with an aim of eliciting the broadest neutralizing antibody titers possible against HIV-1. Methods to chemoselectively and genetically control Env spike tethering so as to minimally affect broadly neutralizing epitopes will also be explored. Besides application to vaccine development, chemical tethering of HIV-1 spikes is expected to enhance knowledge of Env structure-function and provide new avenues for biophysical studies.